Method for the enzymatic preparation of aromatic substances

ABSTRACT

A method for preparing aromatic substances having formula (I), wherein a substrate of formula (II) or an isomer of these precursors, also termed a substrate, in particular selected from those of formula (III), in which formulae R 1  may be a --CH3, --CH2OH, --CHO, --COOH, or (a) radical, R 2  may be a --H, --OH or --O--CH3 radical R 3  may be a --H, --OH or --O--CH3 radical, and R 2  and R 3  may form a methylenedioxy bridge (b), is exposed to the activity of a lipoxygenase. ##STR1##

This application is a continuation of application Ser. No. 08/211,036 filed as PCT/FR93/00745 Jul. 21, 1993, now abandoned.

The subject of the invention is a method for the enzymatic preparation of some aromatic substances.

Aromatic substances is understood to refer to all substances used for the perfume which they release and/or the taste which they provide in the food, pharmaceutical or perfume industry.

The method in accordance with the invention makes it possible to prepare aromatic substances of formula: ##STR2## in which

R₂ may be a radical --H, --OH, --O--CH₃,

R₃ may be a radical --H, --OH, --O--CH₃,

R₂ and R₃ may form a methylenedioxy bridge ##STR3##

These aromatic substances advantageously belong to the group comprising: vanillin, benzaldehyde, anisaldehyde, para-hydroxybenzaldehyde, heliotropin and protocatechnic aldehyde.

BACKGROUND OF THE INVENTION

It was hitherto known to prepare vanillin according to various processes:

chemically from guaiacol, but the vanillin is obtained in a mixture containing numerous impurities, because of which the yields are average;

from the degradation of lignin, but obtaining vanillin in the pure state requires large consumption of chemical products and energy, and results in numerous ecologically toxic waste products;

from safrole, a compound present in essence of sassafras, thus making it possible to obtain an isovanillin/vanillin mixture, two isomers which are difficult to separate;

from eugenol, a compound present in oil of cloves, this compound being subjected to a first step of isomerization to isoeugenol (cis and trans in variable proportions) and then a second step of oxidation to give vanillin; however, this chemical process gives very average yields.

In addition, for all the processes of the prior art resorting to at least one chemical manipulation, the product obtained can only be called "synthetic or semisynthetic vanillin" and can in no way be called "vanillin of natural origin", even if the starting substrate is natural (the case of eugenol or safrole).

Regarding benzaldehyde, it is produced, for example, from cinnamaldehyde treated in alkaline medium under pressure and at high temperature (U.S. Pat. No. 4,617,419).

As regards anisaldehyde, it is conventionally produced by chemical oxidation of anethole, which is found in large quantities in essence of star anise.

However, just as for vanillin and for the same reasons, these two compounds cannot be qualified as "natural".

SUMMARY OF THE INVENTION

The aim of the invention is to overcome the disadvantages of the prior art and it proposes a biotechnological method which makes possible the preparation of some aromatic substances of good purity and with high yields.

Thus, the Applicant has had the merit of establishing that it was possible to prepare aromatic substances of formula: ##STR4## by subjecting one of their precursors, called the substrate, corresponding to the formula: ##STR5## or one of the isomers of these precursors, also called substrate, chosen especially from those corresponding to the formula: ##STR6## in which

R₁ may be a radical --CH₃, --CH₂ OH, --CHO, --COOH, ##STR7##

R₂ may be a radical --H, --OH, --O--CH₃,

R₃ may be a radical --H, --OH, --O--CH₃,

R₂ and R₃ may form a methylenedioxy bridge ##STR8## to the action of an enzyme: lipoxygenase or lipoxidase.

This enzyme is itemized under the reference EC 1.13.11.12 in the work "Enzyme Nomenclature" (IU BMB Academic Press 1992).

It is naturally present in numerous plants such as, for example, soya, wheat or beet and in certain animal organs such as, for example, pig, calf or fish livers. These plant or animal materials may thus constitute industrial sources of this enzyme which, in addition, may also be synthesized by certain microorganisms.

Lipoxygenase is known in particular for its participation in the first step of a process making it possible to synthesize aldehydes from fatty acids. In fact, its oxidative catalytic activity on unsaturated fatty acids, and more particularly on linoleic and linolenic acids, gives rise to the formation of a hydroperoxide which may subsequently be cleaved to an oxo acid and an aldehyde by a hydroperoxide lyase.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the kinetics of production of vanillin from isoeugenol by lipoxygenase.

FIG. 2 illustrates the production of vanillin by lipoxygenase as a function of the concentration of isoeugenol.

FIG. 3 illustrates the production of vanillin from isoeugenol as a function of the concentration of lipoxygenase.

DESCRIPTION OF A PREFERRED EMBODIMENT

Now, within the context of the invention, it has been found that lipoxygenase, whatever its origin, could by itself make possible the preparation of aromatic substances of formula: ##STR9## from substrates represented by the abovementioned formulae and according to the following reaction: ##STR10##

(R₁, R₂ and R₃ having the meanings given above).

The preferred aromatic substances prepared according to the method in accordance with the invention are summarized in Table I.

                                      TABLE I                                      __________________________________________________________________________                                               Product obtained by                                                   Name of a possible                                                                      subjecting the sub-                  Substituents of the substrate subjec-                                                                           isomer of the sub-                                                                      strate or its isomer                 ted to the action of the lipoxygenase                                                                  Name of the cor-                                                                        strate according to                                                                     to the action of the                 R.sub.1     R.sub.2                                                                              R.sub.3                                                                              responding substrate                                                                    the invention                                                                           lipoxygenase                         __________________________________________________________________________     CH.sub.3    OCH.sub.3                                                                            OH    isoeugenol                                                                              eugenol  vanillin                             CH.sub.2 OH OCH.sub.3                                                                            OH    coniferyl alcohol vanillin                             CHO         OCH.sub.3                                                                            OH    coniferyl aldehyde                                                                               vanillin                             COOH        OCH.sub.3                                                                            OH    ferulic acid      benzaldehyde                         CH.sub.2 OH H     H     cinnamyl alcohol  benzaldehyde                         CHO         H     H     cinnamaldehyde    benzaldehyde                         COOH        H     H     cinnamic acid     benzaldehyde                         CH.sub.3    H     OCH.sub.3                                                                            anethole estragol anisaldehyde                         CH.sub.3    H     OH    anol     chavicol para hydroxyben-                                                               zaldehyde                            CH.sub.3                                                                                    ##STR11##  isosafrole                                                                              safrole  heliotropine                         COOH        OH    OH    caffeic acid      protocatechnic al-                                                             dehyde                                ##STR12##  OCH.sub.3                                                                            OH    curcumin          vanillin                             __________________________________________________________________________

Eugenol, chavicol, estragol and safrole, which are the isomers of isoeugenol, anol, anethole and isosafrole respectively, may themselves be converted by lipoxygenase to the corresponding aldehyde, namely vanillin, hydroxybenzaldehyde, anisaldehyde and heliotropin, but with a lower yield.

In fact, the most important condition for lipoxygenase to be able to exert its action is that the substrate contains a correctly positioned unsaturated bond: this is especially the case for all the molecules represented by the formulae corresponding to the "substrate" for which the unsaturated bond situated on the chain bearing R₁ or CH₂ is either in the alpha or in the beta position of the aromatic ring. This is also the case for the known examples represented by linoleic and linolenic acids, in which either their carbon 9 or their carbon 13 (constituting a non-conjugated cis-cis diene system) is the site of a peroxidation.

The method in accordance with the invention thus consists in placing together, in a stirred and thermo-regulated buffered medium, a lipoxygenase and a substrate corresponding to one of the formulae represented above for a sufficient time to allow the preparation of the corresponding aromatic substance, it being possible for the latter to be subsequently recovered by standard solvent extraction or distillation techniques.

In the method in accordance with the invention, lipoxygenase may be used in purified or unpurified form.

Advantageously:

when it is purified, that is to say when it generally has an activity greater than 30,000 u/mg sic!, (1 unit corresponding to an increase in the absorbance at 234 nmof a value of 0.001 per minute when linoleic acid is the substrate, at pH 9 and at 25° C.), lipoxygenase is used in an amount of from 1 to 500 mg per gram of substrate, preferably from 5 to 100 mg per gram of substrate and even more preferably from 10 to 50 mg per gram of substrate, (by way of example, a lipoxygenase marketed by SIGMA in the form of an enzymatic preparation containing at least 60% of proteins may be used), and

when it is not purified, that is to say when it contains less than 7,500 U/mg, as is the case in commercial acetone powders (preconcentrated form from a raw material), or in ground fresh plant material or in a plant material flour, or, alternatively, in a ground fresh animal organ such as the liver, it will be used in an amount of 0.01 to 50 g per gram of substrate, preferably from 0.025 to 10 g per gram of substrate and even more preferably from 0.1 to 5 g per gram of substrate.

According to the method in accordance with the invention, the lipoxygenases preferably used are soybean lipoxygenase and wheatgem lipoxygenase.

According to an advantageous embodiment of the method in accordance with the invention, the concentration of the substrate for the lipoxygenase is established between 2.5 and 100 g per liter of reaction medium, and preferably between 20 and 75 g per liter of reaction medium.

In accordance with the invention, this reaction medium is a buffered aqueous medium. The buffers used are standard buffers such as potassium phosphate buffer and/or sodium phosphate buffer and/or sodium borate buffer and/or sodium borate/citrate/phosphate buffer.

The molarities of these buffers are advantageously between 0.05M and 0.5M and their pH is between 2 and 10 and preferably between 5 and 10. Adjustments to pH are made either with aqueous potassium or sodium hydroxide solutions or with phosphoric acid.

According to other advantageous characteristics of the invention:

the temperature of the reaction medium varies between 4° and 40° C., and preferably between 22° and 32° C.;

the duration of the reaction varies between 30 minutes and 7 days, preferably between 2 hours and 5 days, and is determined by monitoring the reaction kinetics;

the stirring must be sufficiently vigorous to homogenize or emulsify the whole of the reaction medium and to oxygenate all of the substrate present, and is carried out at a speed between 150 and 250 rev/min.

In addition, an emulsifying agent such as TWEEN 80 may be introduced into the medium in a proportion of 0.05 to 2% by volume relative to the reaction medium.

According to an advantageous embodiment of the method in accordance with the invention, depending on the substrate used, and in particular when the substrate is a compound of phenolic nature, due to the toxicity of phenols towards the enzyme, it may be necessary to add to the reaction medium at least one adsorbing agent or one complexing agent chosen from the group comprising activated charcoal at a concentration between 5 and 50 g per liter, the latter known under the trade name "Amberlite" of type XAD₂ at a concentration between 5 and 50 g per liter, beta-cyclodextrin at a concentration between 3 and 18 g per liter and polyvinylpolypyrrolidone (PVPP) at a concentration between 10 and 60 g per liter, so as to protect the said enzyme.

The invention is not strictly limited to the described method; on the contrary, it embraces all the variants thereof. The Applicant has, for example, conclusively investigated the conversion of beta-carotene to beta-ionone by the action of lipoxygenase, which in this case acts on an unsaturated bond located in a gamma position relative to the ring of the molecule, conjugated with an unsaturated bond located in an alpha position relative to the same ring.

The examples which follow will make possible a better understanding of the invention; they are not limiting, and relate to the advantageous embodiments of the invention.

EXAMPLE 1

To a flask containing 20 ml of sodium borate buffer having a molarity of 0.1M and a pH of 6.5 are added 0.75 g of isoeugenol, 20 mg of lipoxygenase originating from soybean and assaying at 36,000 U/mg and 0.05 g of TWEEN 80.

After incubation for three days at 28° C. and with stirring at a speed of 150 rev/min, the whole test sample is extracted with dichloroethane and the vanillin is subsequently assayed by HPLC.

The vanillin production is 2.85 g/l.

EXAMPLE 2

Under the same conditions as in Example 1, 0.75 g of eugenol is added to the reaction medium in the place of isoeugenol in the presence of 0.5 g of activated charcoal.

The vanillin yield is 28.5 mg/liter.

EXAMPLE 3

Eight flasks containing the same ingredients as in Example 1 are incubated at 28° C. on a stirrer table revolving at 150 rev/min. Each day a sample is extracted with dichloroethane and assayed by HPLC.

The kinetics are reproduced in FIG. 1.

The vanillin production reaches 2.82 g/l.

EXAMPLE 4

Under the same conditions as in the above examples, the concentrations of isoeugenol are varied from 5 to 100 g/l of reaction medium.

A vanillin production, represented in FIG. 2, which reaches 2.2 g/l is obtained.

EXAMPLE 5

Under the conditions described in Example 1, the concentration of lipoxygenase added to the reaction medium is varied from 0 to 2.5 g/l of reaction medium.

The corresponding amounts of vanillin produced are represented in FIG. 3.

The vanillin production reaches 2.9 g/l.

EXAMPLE 6

Under the same conditions as in Example 1, 10 g/l of curcumin are added, treated beforehand with potassium hydroxide in order to solubilize it in the buffer medium.

Vanillin is obtained in a yield of 37 mg/l.

EXAMPLE 7

Under the same conditions as in Example 1, isoeugenol is replaced by 2 g/l of coniferyl alcohol.

In two days, vanillin is obtained in a yield of 240 mg/l.

EXAMPLE 8

Under the same conditions as in Example 1, 25 g/l of cinnamaldehyde are added.

After incubation for four days, 1.1 g/l of benzaldehyde are detected in the medium.

EXAMPLE 9

Under the same conditions as in Example 1, 8 g/l of anethole are added.

After four days, 448 mg/l of anisaldehyde are measured.

EXAMPLE 10

To a flask containing 25 g of ground eggplant and 25 g of sodium borate buffer having a molarity of 0.1M and a pH of 6.5 are added 0.4 g of isoeugenol and 0.05 g of TWEEN 80. After incubation for 3 days at 28° C. and with stirring at a speed of 200 rev/min, 1.75 g/l of vanillin are detected.

EXAMPLE 11

To a flask containing 20 g of sodium borate buffer having a molarity of 0.1M and a pH of 6.5 and 0.75 g of isoeugenol are added 5 g of ground fresh pig liver and 0.05 g of TWEEN 80. After incubation for 2 days at 28° C. and with stirring at a speed of 200 rev/min, the amount of vanillin measured is 0.7 g/l. 

We claim:
 1. Method for the preparation of aromatic substances having formula I: ##STR13## wherein a substrate selected from the group consisting of eugenol, estragol, chavicol, safrole, compounds having the formula II: ##STR14## and compounds having the formula III: ##STR15## in which R₂ is a radical selected from the group consisting of --CH₃, -- CH₂ OH, --CHO, --COOH, or ##STR16## R₂ is a radical selected from the group consisting of --H, --OH, or --O--CH₃,R₃ is a radical selected from the group consisting of --H, --OH, or --O--CH₃,with the proviso that when R₂ and R₃ represent together --OH, then R₁ does not represent --COOH, or R₂ and R₃ form a methylenedioxy bridge, is reacted with a lipoxygenase, and the aromatic substances having formula I are recovered, and wherein said lipoxygenase is used in purified form in an amount of 1 to 500 mg per gram of substrate or in unpurified form in an amount of 0.01 to 50 g per gram of substrate.
 2. Method according to claim 1, wherein aromatic substances having formula I are selected from the group consisting of vanillin, benzaldehyde, anisaldehyde, parahydroxybenzaldehyde, heliotropin and protocatechic aldehyde.
 3. Method according to claim 1, wherein the lipoxygenase in purified form is used in an amount of 5 to 100 mg per gram of substrate.
 4. Method according to claim 1, wherein the lipoxygenase in purified form is used in an amount of 10 to 50 mg per gram of substrate.
 5. Method according to claim 1, wherein the lipoxygenase in unpurified form is used in ah amount of 0.025 to 10 g per gram of substrate.
 6. Method according to claim 1, wherein the lipoxygenase in unpurified form is used in an amount of 0.1 to 5 g per gram of substrate.
 7. Method according to claim 1, wherein a lipoxygenase or a source of lipoxygenase of plant origin is used.
 8. Method according to claim 1, wherein a lipoxygenase or a source of lipoxygenase of animal origin is used.
 9. Method according to claim 1, wherein the concentration of the substrate is between 2.5 and 100 g per liter of reaction medium.
 10. Method according to claim 1, wherein the concentration of the substrate for the lipoxygenase is established between 20 and 75 g per liter of reaction medium.
 11. Method according to claim 1, wherein the reaction medium is a buffered medium having a pH between 2 and
 10. 12. Method according to claim 1, wherein the reaction medium is a buffered medium having a pH between 5 and
 10. 13. Method according to claim 1, wherein the temperature of the reaction medium is between 4° and 40° C.
 14. Method according to claim 1, wherein the temperature of the reaction medium is between 22° and 32° C.
 15. Method according to claim 1, wherein the duration of the reaction varies from 30 minutes to 7 days.
 16. Method according to claim 1, wherein the duration of the reaction varies from 2 hours to 5 days.
 17. Method according to claim 1, wherein the reaction medium is stirred at a speed between 150 and 250 rev/min.
 18. Method according to claim 1, wherein an emulsifying agent is added in an amount of 0.05 to 2%, by volume relative to the reaction medium.
 19. Method according to claim 1, wherein at least one adsorbing agent or one complexing agent is added to the reaction medium when a compound wherein R₂ or R₃ or both are OH is used as substrate.
 20. Method according to claim 19, wherein the adsorbing agent or the complexing agent is selected from the group consisting of activated charcoal at a concentration between 5 and 50 g per liter, Amberlite® of type XAD₂ at a concentration between 5 and 50 g per liter, B-cyclodextrin at a concentration between 3 and 18 g per liter, and polyvinylpolypyrrolidone at a concentration between 10 and 60 g per liter. 